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Industrial Crops & Products
journal homepage:www.elsevier.com/locate/indcropPhenolic compounds pro
file, nutritional compounds and bioactive
properties of Lycium barbarum L.: A comparative study with stems and fruits
Tânia C.S.P. Pires
a,b, Maria Inês Dias
a, Lillian Barros
a,⁎, Ricardo C. Calhelha
a, Maria José Alves
a,
Celestino Santos-Buelga
b, Isabel C.F.R. Ferreira
a,⁎aCentro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal
bGrupo de Investigación en Polifenoles (GIP-USAL), Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, 37007 Salamanca, Spain
A R T I C L E I N F O Keywords: Lycium barbarum L. Fruits/stems Nutritional value Phenolic composition Bioactive properties A B S T R A C T
The increasing awareness of the possible health benefits of berry fruits (Lycium barbarum L.) has led to a higher consumption of this type of food products. One of the many examples are the fruits from Lycium genus, tradi-tionally used due to their beneficial properties and health benefits associated with liver, kidney, eyesight, im-mune system, circulation and longevity disorders. In the present study fruits and stems of Lycium barbarum L. (goji) were characterized in terms of nutritional profile, sugars, organic acids, fatty acids and tocopherols. Furthermore, a phenolic characterization of their hydromethanolic extracts was performed and correlated with bioactive properties such as antioxidant, hepatotoxic and antibacterial activities. Stems presented higher values of energy, MUFA (monounsaturated fatty acids), tocopherols andflavonols. Stems also showed greater anti-oxidant and antibacterial (against Gram-negative bacteria) activities. Otherwise, fruits revealed higher contents of sugars, PUFA (polyunsaturated fatty acids) and hydroxycinnamic acid derivatives, and greater activity against Gram-positive bacteria. This is an innovative study that shows the high potential of goji stems and fruits as sources of bioactive compounds, which could be used in nutraceutical formulations, or incorporated into food products with functional properties. Furthermore, the use of stems could bring industrial sustainability as a valuable by-product, which has been scarcely reported.
1. Introduction
The interest in many traditional herbs and plant food supplements, as a source of nutritional antioxidants, is due to the increasing knowl-edge of the role of antioxidants and free radicals in human health (Dahech et al., 2013). The consumption of plants belonging to the Ly-cium genus has increased exponentially, not only due to their tradi-tionally usage in Chinese medicine, but also because of their wide ac-ceptance as food ingredients (Dahech et al., 2013;Dong et al., 2009). The berries are commonly consumed in soups, as porridge with rice and added to numerous meat and vegetable dishes (Potterat and Food, 2010), eaten raw, as a juice, wine or in tea preparations, as also pro-cessed as tinctures, powders, and tablets (Potterat and Food, 2010).
One of the most widely studied species of this genus is Lycium barbarum L., which has several vernacular names, being“goji” the most common one (Amagase and Farnsworth, 2011). Since the beginning of the 21th century, goji products have been introduced in Europe and North America and their consumption has increased rapidly due to their claimed beneficial properties for wellbeing and longevity (D’Amato
et al., 2013). Goji berries have been associated with a wide range of health benefits, including the treatment of diseases related to liver, kidney, eyesight, immune system, circulation and longevity, as also with sexual activity (Tang et al., 2012). Recent studies also suggest that L. barbarum leaves have shown a broad development and application prospects in the food industry due to the rich nutrients, biological active ingredients and trace elements (Liu et al., 2012).
The interest in the composition of berry fruits has been also in-tensified because of an increased awareness of their possible health benefits, as they are rich sources of micronutrients and phytochemicals such as polyphenols. Some of these phenolic compounds, which can act as antioxidants and antimicrobials, have been identified by different authors (Amagase and Farnsworth, 2011;Dahech et al., 2013;Liu et al., 2017), but to the authors’ best knowledge there is no previous in-formation about the chemical and bioactive characteristics of L. bar-barum stems. The present study describes and compares the nutritional and chemical composition of Lycium barbarum L. stems and fruits; moreover, a phenolic characterization of its hydromethanolic extracts was performed and correlated with bioactive properties (e.g.,
https://doi.org/10.1016/j.indcrop.2018.06.046
Received 8 February 2018; Received in revised form 5 May 2018; Accepted 8 June 2018 ⁎Corresponding authors.
E-mail addresses:[email protected](L. Barros),[email protected](I.C.F.R. Ferreira).
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
antioxidant, hepatotoxic and antimicrobial). The results of this study might be useful to maximize the potential of stems as by-products with functional properties with interest in food and pharmaceutical in-dustries.
2. Materials and methods 2.1. Standards and reagents
Acetonitrile (99.9%) was of HPLC grade from Fisher Scientific (Lisbon, Portugal). Phenolic standards were from Extrasynthèse (Genay, France). Sulforhodamine B, trypan blue, trichloroacetic acid (TCA), tris (hydroxymethyl)aminomethane (Tris), Trolox (6-hydroxy-2,5,7,8-tet-ramethylchroman-2-carboxylic acid) and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhy-drazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). p-Iodonitrotetrazolium chloride (INT) was from Panreac Applichem (Barcelona, Spain), Tryptic Soy Broth (TSB) and Mueller-Hinton (MH) were purchased from Biolab®(Hungary). All other general laboratorial reagents were from Panreac Química S.L.U. (Barcelona, Spain). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, USA).
2.2. Samples
The dried fruits and stems of Lycium barbarum L. were supplied by the company RBR foods from Castro Daire (Portugal). After reception, the dried fruits and stems were reduced to afine dried powder (∼20 mesh) and mixed to obtain homogenate samples.
2.3. Nutritional value of L. barbarum fruits and stems 2.3.1. Proximate composition and energetic value
The dehydrated and powdered fruits and stems were analysed for proteins, fat, carbohydrates and ash according to the AOAC (Association of Official Analytical Chemists) procedures (AOAC, 2016). The AOAC 991.02 was followed to determine the crude protein content (N × 6.25, macro-Kjeldahl method); AOAC 989.05 was used to de-termine crude fat (Soxhlet apparatus with petroleum ether as extraction solvent); AOAC 935.42 was used for ash content determination (in-cineration at 550 ± 15 °C). The total carbohydrates (includingfiber) were calculated by difference, according with the equation: Total car-bohydrates (g/100 g) = 100− (g fat + g protein + g ash). Total energy was calculated according to the following equation: Energy (kcal/ 100 g) = 4 × (g proteins + g carbohydrates) + 9 × (g fat).
2.3.2. Fatty acids
Fatty acids were determined by using a Soxhlet extraction of the dehydrated and powdered fruits and stems in order to obtain a lipidic fraction and after a trans-esterification process, being further analysed by gas chromatography coupled with aflame ionization detector (GC-FID; DANI model GC 1000 instrument, Contone, Switzerland), ac-cording to the procedure previously described by the authors (Dias et al., 2015). The results were expressed in relative percentage of each fatty acid.
2.3.3. Soluble sugars
Soluble sugars were determined in the dehydrated and powdered fruits and stems following a procedure previously described by the authors (Dias et al., 2015). A High performance liquid chromatography system coupled to a refraction index detector (HPLC-RI; Knauer, Smartline system 1000, Berlin, Germany) was used to identify and quantify the soluble sugars. The quantification was performed using the internal standard (melezitose) and the results were expressed in g per 100 g of fruits and stems dry weight.
2.3.4. Organic acids
The dehydrated and powdered fruits and stems were analysed for its organic acids following the procedure previously described by the au-thors (Dias et al., 2015), using an ultra-fast liquid chromatography coupled to photodiode array detector (UFLC-PDA; Shimadzu Copera-tion, Kyoto, Japan). The quantification was performed by comparison of the peak area recorded at 215 nm as preferred wavelength. For quantitative analysis, a calibration curve with known concentration (10− 0.0078 mg/mL) for each available organic acid, was constructed based on the UV signal: oxalic acid (y = 45,973 + 9 × 106x,
R2= 0.9901); quinic acid (y = 46,061 + 610607x, R2= 0.9995); malic acid (y = 92,665 + 912441x, R2= 0.999); citric acid
(y = 45,682 + 1×106x, R2= 0.9997), and succinic acid
(y = 50,689 + 592888x, R2= 0.9996). The results were expressed in g per 100 g of fruits and stems dry weight.
2.3.5. Tocopherols
Tocopherols (four vitamers) were determined according with the procedure previously described by the authors (Dias et al., 2015), in the dehydrated and powdered fruits and stems, by HPLC (Knauer, Smart-line system 1000, Berlin, Germany) coupled to afluorescence detector (FP-2020; Jasco, Easton, MD, USA). For the quantification, an internal standard (tocol) was used, based on thefluorescence signal response of each standard. The results were expressed in mg per 100 g of fruits and stems dry weight.
2.4. Analysis of phenolic compounds
2.4.1. Preparation of the hydromethanolic extracts
To prepare the hydromethanolic extracts, 1 g of each dehydrated and powdered sample was extracted with a methanol/water mixture (80:20, v/v), at 25 °C and 150 rpm, during 1 h, followed byfiltration through a Whatmanfilter paper No. 4. The remaining residue was re-extracted with one additional portion of the methanol/water mixture, and the combined extracts were evaporated under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland) to remove the methanol; then the extracts were frozen, lyophilized and stored at −5 °C for further analysis.
2.4.2. Phenolic compounds
The hydromethanolic extracts were re-dissolved at a concentration of 5 mg/mL in methanol/water (80:20, v/v). The analysis was per-formed using a LC-DAD-ESI/MSn (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) as previously described by (Bessada et al., 2016). The detection was performed using 280, 330 and 370 nm as preferred wavelengths for DAD and in a mass spectrometer equipped with an ESI source and performed in negative mode (Linear Ion Trap LTQ XL mass spectrometer, Thermo Finnigan, San Jose, CA, USA). The identification of the phenolic compounds was performed based on its chromatographic behaviour and UV–vis and mass spectra by compar-ison with available standard compounds, and data reported in the lit-erature giving a tentative identification. For quantitative analysis, a calibration curve with known concentration (200–5 μg/mL) for each available phenolic standard: caffeic acid (y = 406,369 + 388345x, R2= 0.9949); catequin (y =−23,200 + 84950x, R2= 0.9999);
chlorogenic acid (y =−161,172 + 168823x, R2= 0.9999); ferulic acid (y =− 185,462 + 633126x, R2= 0.9999); kaempferol-3-O-ruti-noside (y = 30,861 + 11117x; R2= 0.9998); p-coumaric acid
(y = 6966.7 + 301950x, R2= 0.9999); quercetin-3-O-rutinoside
(y = 76,751 + 13343x, R2= 0.9998); quercetin-3-O-glucoside (y =−160,173 + 34843x, R2= 0.9998); sinapic acid
(y = 30,036 + 197337x, R2= 0.9997), was constructed based on the
UV signal. For the identified phenolic compounds for which a com-mercial standard was not available, the quantification was carried out through the calibration curve of the most similar available standard. The results were expressed as mg per g of extract.
2.5. Evaluation of bioactive properties 2.5.1. Antioxidant activity
The hydromethanolic extracts were re-dissolved in methanol:water (80:20, v/v) to obtain stock solutions of 2.5 mg/mL, which were further diluted to obtain a range of concentrations (2.5 mg/mL to 0.07 mg/mL) for antioxidant activity evaluation by DPPH radical-scavenging, redu-cing power, inhibition ofβ-carotene bleaching, and TBARS inhibition assays (Barros et al., 2013). Thefinal results were expressed as EC50
values (mg/mL), which means sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay. Trolox was used as a positive control.
2.5.2. Antibacterial activity
The hydromethanolic extracts were re-dissolved in water in order to obtain stock solutions of 100 mg/mL, and then submitted to further dilutions. The microorganisms used were clinical isolates from patients hospitalized in various departments of the Local Health Unit of Bragança and Hospital Center of Trás-os-Montes and Alto-Douro Vila Real, Northeast of Portugal. Seven Gram-negative bacteria (Escherichia coli, E. coli ESBL (extended spectrum of beta-lactamase), Klebsiella pneumoniae, K. pneumoniae ESBL, Morganella morganii, Pseudomonas aeruginosa and Acinetobacter baumannii, isolated from urine and ex-pectoration) and five Gram-positive bacteria (MRSA- methicillin-re-sistant Staphylococcus aureus, MSSA- methicillin-susceptible Staphylococcus aureus, Staphylococcus aureus, Listeria monocytogenes and Enterococcus faecalis) were used to screen the antibacterial activity. Minimum inhibitory concentrations (MIC) were determined by the microdilution method and the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay was used by following the methodology pro-posed byKuete et al. (2011a,b)with some modifications (Dias et al., 2016). The antibiotic susceptibility profile was obtained for all the tested bacteria (Table A1, Supplementary material). MIC was defined as the lowest concentration that inhibits the visible bacterial growth. 2.5.3. Hepatotoxicity
The hydromethanolic extracts were re-dissolved in water to obtain stock solutions of 4 mg/mL, and then submitted to further dilutions. For hepatotoxicity evaluation, a porcine liver cells primary culture (PLP2) was prepared from a freshly harvested porcine liver obtained from a local slaughterhouse, according to a procedure established by the au-thors (Abreu et al., 2011). Ellipticine was used as positive control and the results were expressed in GI50values, concentration that inhibited
50% of the net cell growth. 2.5.4. Statistical analysis
Three samples were used for each plant part and all the assays were carried out in triplicate. The results were expressed as mean values and standard deviation (SD), being analysed using a Student’s t-test, with α = 0.05. Furthermore, a Pearson’s correlation analysis between the bioactivities and the different groups of phenolic compounds (sum of phenolic acids, sum offlavan-3-ols, sum of flavonols and sum of phe-nolic compounds) was carried out, with a 95% confidence level. The analyses were carried out using IBM SPSS Statistics for Windows, Version 22.0. (IBM Corp., Armonk, New York, USA).
3. Results and discussion 3.1. Nutrient composition
Data on the proximate composition and energetic value of L. bar-barum are shown inTable 1. Carbohydrates were the most abundant macronutrients in fruits and stems (87 and 78.1 g/100 g dw, respec-tively). Stems presented the highest contents of ash, proteins and fat (9.9, 7.4, and 4.6 g/100 g dw, respectively), while fruits presented proteins as the second major macronutrient (5.3 g/100 g dw), followed
by fat and ash (4.1 and 3.21 g/100 g dw, respectively).Yan et al. (2014) reported different results for goji fruits from China, describing a higher content of proteins and fat (12.1 and 6.89 g/100 g dw, respectively) and a lower ash content (0.95 g/100 g dw). These differences might be ex-plained by the cultivar and/or different edaphic conditions that can lead to variable nutritional contents.
Soluble sugars and organic acids of the studied fruits and stems are also presented inTable 1. Fructose, glucose and sucrose were the only forms of monosaccharides detected in fruits and stems, being glucose the most abundant one, followed by fructose and sucrose. As expected, fruits presented much higher content in soluble sugars (27.9 g/100 g dw) than stems (1.08 g/100 g dw). These results are in agreement with those obtained byMikulic-Petkovsek et al. (2012)in goji fruits from Slovenia, where glucose and fructose were also the prevailing sugars detected, although those authors reported a higher total sugars content. Regarding organic acids (Table 1), fruits and stems presented very different profiles, however, no statistically significant differences were found in the sum of the organic acids between samples. Citric, succinic and oxalic (1.29, 0.77, and 0.010 g/100 g dw, respectively) acids were detected in the fruit; while malic, oxalic and quinic (0.899, 0.65, and 0.53 g/100 g dw, respectively) acids were found in the stems. Oxalic acid was the only organic acid common in both samples.Donno et al. (2015), in goji fruits from Italy, reported the presence of several organic acids, including malic, quinic and tartaric acids that were not detected in samples of this study. These differences might be due to the physical state of the samples and/or the extraction method, as Donno et al. (2015)analysed the organic acids in fresh or semi-fresh samples (stored at 4 °C for a few days) and using ethanol as extraction solvent.
Fatty acids were also determined in fruits and stems of goji fruits and the results are shown inTable 2. Sixteen fatty acids were identified in the fruits, being polyunsaturated fatty acids (PUFA) the predominant group, mainly due to the presence of linoleic acid (C18:2n6, 53.4%), followed by oleic acid (C18:1n9, 16.5%) and palmitic acid (C16:0, 12.77%). Similar results were obtained byYan et al. (2014)in goji fruits from China, that described linoleic acid (66.81%) and oleic acid (17.13%) as the major fatty acids. In stem samples, eighteen fatty acids were identified, being saturated fatty acids (SFA) predominant, espe-cially palmitic (C16:0, 15.94%) and lignoceric acids (C24:0, 15.3%), followed by linolenic acid (C18:3n3, 14.8%).
Regarding tocopherols (Table 2), both samples presented only two vitamers. The highest content of tocopherols (3.59 mg/100 g dw) was Table 1
Proximate composition, soluble sugars and organic acids in fruits and stems of Lycium barbarum L. (mean ± SD).
Fruits Stems t-Students test
p-value Nutritional value (g/100 g dw) Fat 4.1 ± 0.3 4.6 ± 0.3 0.040 Proteins 5.3 ± 0.2 7.4 ± 0.2 < 0.001 Ash 3.21 ± 0.02 9.9 ± 0.1 < 0.001 Total carbohydrates 87 ± 6 78.1 ± 0.4 < 0.001
Energy contribution (kcal/ 100 g dw) 408 ± 1 383 ± 2 < 0.001 Soluble sugars (g/100 g dw) Fructose 12.7 ± 0.4 0.45 ± 0.01 < 0.001 Glucose 14.4 ± 0.4 0.42 ± 0.01 < 0.001 Sucrose 0.8 ± 0.1 0.21 ± 0.02 < 0.001 Sum 27.9 ± 0.9 1.08 ± 0.05 < 0.001 Organic acids (g/100 g dw) Oxalic acid 0.010 ± 0.001 0.65 ± 0.001 < 0.001 Quinic acid nd 0.53 ± 0.03 – Malic acid nd 0.899 ± 0.004 – Citric acid 1.29 ± 0.02 nd – Succinic acid 0.77 ± 0.07 nd – Sum 2.07 ± 0.01 2.08 ± 0.03 0.677
determined in the stems, mainly due to the presence of α-tocopherol (3.37 mg/100 g dw), with minor levels ofβ-tocopherol (0.22 mg/100 g dw). Significant lower concentrations of tocopherols were found in the fruits, also containing α-tocopherol, but with δ-tocopherol as the second vitamer (0.23 and 0.09 mg/100 g dw, respectively). To the au-thors’ best knowledge, there are no previous studies of tocopherols composition in goji fruits and stems.
3.2. Individual phenolic profile
The peak characteristics (retention time, wavelength of maximum absorption and mass spectral data), tentative identification and quan-tification of phenolic compounds present in the hydromethanolic ex-tracts of L. barbarum fruits and stems are presented inTable 3. An ex-emplificative phenolic profile of the hydromethanolic extracts of both types of samples, recorded at 280 nm, is shown inFig. 1. Fruits and stems presented different phenolic profile, with the presence of hy-droxybenzoic (galloyl derivatives) and hydroxycinnamic (caffeic, p-coumaric, ferulic and sinapic acid derivatives) acid derivatives, flavan-3-ols, and flavonols (quercetin and kaempferol derivatives). Sixteen compounds were identified in fruit samples: eight flavonols (peaks 6, 16, 17, 18, 19, 20, 21, and 22), seven phenolic acid derivatives (peaks 1, 2, 3, 4, 7, 10, and 14), and oneflavan-3-ol (peak 9), while eleven compounds were detected in the stems, most of which were phenolic acid derivatives (peaks2, 4, 5, 7, 8, 11, 12, and 13), together with two flavonols (peaks 16 and 20) and one flavan-3-ol (peak 15). Only three chlorogenic acids (peaks2, 4 and 7) were common to both samples. Peaks7, 9, 12, 14, 16, 19, and 20 (5-O-caffeoylquinic acid, catechin,
caffeic acid, p-coumaric acid, rutinoside, quercetin-3-O-glucoside, and kaempferol-3-O-rutinoside, respectively) were identified by its UV and mass spectra, and retention characteristics in comparison with commercial standards. Compounds19 and 20 had been previously reported by other authors in goji leaves (Mocan et al., 2017) and fruits (Bondia-Pons et al., 2014;Inbaraj et al., 2010).
Flavonols were the most abundant phenolic compounds in goji stems, although mostly due to the presence of quercetin-3-O-rutinose (rutin, peak16), with minor levels of kaempferol-3-O-rutinose (peak 20). The presence of rutin as a majorflavonol in different parts of goji plants has been consistently reported by several authors (Affes et al., 2017; Bondia-Pons et al., 2014; Mocan et al., 2017, 2015a,b, 2014; Protti et al., 2017;Qian et al., 2004;Zhang et al., 2016). Flavonols were less abundant in the fruits, despite they presented greater variety of these compounds. Quercetin-3-O-glucoside (isoquercitrin, peak19) was positively identified by comparison with a standard. Peak 18 presented the same UV and mass spectral characteristics as compound 19 ([M−H]−at m/z 463), thus corresponding to a quercetin hexoside,
which was tentatively assigned as hyperoside (i.e., quercetin-3-O-ga-lactoside), owing to the previous identification of both isoquercitrin and hyperoside in goji fruits (Lycium spp) byQian et al. (2004) and Donno et al. (2015). This identity is also coherent with its chromato-graphic behaviour, as galactosides are expected to elute before its corresponding glucosides (Santos-Buelga et al., 2003). Peak 17 pre-sented a pseudomolecular [M−H]− at m/z 447 releasing an MS2
fragment at m/z 301, allowing its identification as a quercetin-deox-yhexoside, tentatively associated to quercitrin (quercetin-3-O-rhamno-side) previously reported in different goji samples (Mocan et al., 2015a, 2014;Protti et al., 2017;Zhou et al., 2017). Peak6 showed a UV spectra characteristic of a quercetin derivative, and a pseudomolecular ion [M−H]−at m/z 933, yielding fragments at m/z 609 ([M−H-324]−
, loss of two hexosyl units) and m/z 301 ([M−H-308]−, loss of a
ruti-nosyl unit), being tentatively identified as quercetin-dihexoside-ruti-noside. A compound with the same characteristics (rutin di-hexose) was reported in hydromethanolic extracts of goji fruits from Finland (Bondia-Pons et al., 2014). Other twoflavonols derived from kaemp-ferol were also detected in the fruits. As above indicated, peak21 was identified as kaempferol-3-O-glucoside by comparison with a standard, previously reported in goji fruits byAffes et al. (2017)and leaves by Mocan et al. (2017). Peak22 was tentatively assigned as kaempferol-rhamnoside based on its pseudomolecular ion ([M−H]−
at m/z 431) releasing a unique fragment at m/z 285, by analogy with the identi fi-cations made for quercetin glycosides.
Twoflavan-3-ol derivatives were detected in the analysed samples and stems. Catechin (peak9) was positively identified in the fruit by comparison with a commercial standard, whereas peak15, found in the stems, was associated to a procyanidin dimer according to its UV spectrum, pseudomolecular ion ([M−H]−at m/z 577) and MS2
frag-ments at m/z 289, 245 and 203.
The remaining compounds detected in goji samples corresponded to phenolic acid derivatives, most of them derivatives of hydroxycinnamic acids, which were the most abundant compounds in the fruits. Three chlorogenic acids, peaks 2, 4 and 7 showing a pseudomolecular ion [M−H]−
at m/z 353 yielding a main product ion at m/z 191 (depro-tonated quinic acid), were identified as cis and trans 3-O-caffeoylquinic acids and trans 5-O-caffeoylquinic acid, respectively, based on the hierarchical keys previously described byClifford et al. (2003,2005). These type of compounds are among the most common phenolic com-pounds usually reported in goji samples, although most authors do not indicate the particular derivative, but just refer to them as chlorogenic acid or isomers (Affes et al., 2017; Bondia-Pons et al., 2014; Donno et al., 2015;Mocan et al., 2015a,b,2014;Qian et al., 2004;Zhang et al., 2016;Zhou et al., 2017). OnlyMocan et al. (2017)described the pre-sence of different caffeoylquinic acids in the leaves of cultivated L. barbarum from Romania, with particularly high contents of 3-O-caf-feoylquinic acid.Inbaraj et al. (2010)also reported 3-O-caffeoylquinic Table 2
Fatty acids and tocopherols in fruits and stems of Lycium barbarum L. (mean ± SD).
Fruits Stems t-Students test p-value
Fatty acids (relative percentage, %)
C8:0 0.65 ± 0.04 0.60 ± 0.04 0.020 C10:0 0.10 ± 0.01 0.15 ± 0.01 < 0.001 C12:0 0.19 ± 0.02 0.19 ± 0.02 0.442 C14:0 0.38 ± 0.02 1.7 ± 0.1 < 0.001 C14:1 0.37 ± 0.03 0.35 ± 0.02 0.015 C15:0 0.21 ± 0.02 0.29 ± 0.01 < 0.001 C16:0 12.77 ± 0.07 15.94 ± 0.08 < 0.001 C16:1 0.29 ± 0.02 nd – C17:0 0.48 ± 0.05 0.90 ± 0.04 < 0.001 C18:0 7.50 ± 0.06 9.1 ± 0.2 < 0.001 C18:1n9 16.5 ± 0.5 5.12 ± 0.06 < 0.001 C18:2n6 53.4 ± 0.5 9.7 ± 0.2 < 0.001 C18:3n3 1.68 ± 0.02 14.8 ± 0.3 < 0.001 C20:0 1.30 ± 0.07 12.84 ± 0.01 < 0.001 C20:2 nd 1.3 ± 0.2 – C20:3n3 0.35 ± 0.04 0.73 ± 0.04 0.000 C22:0 2.75 ± 0.08 10.4 ± 0.1 < 0.001 C23:0 nd 0.69 ± 0.01 – C24:0 nd 15.3 ± 0.3 – SFA 26.1 ± 0.1 68.0 ± 0.5 < 0.001 MUFA 17.2 ± 0.6 5.46 ± 0.04 < 0.001 PUFA 56.8 ± 0.5 26.6 ± 0.4 < 0.001 Tocopherols (mg/100 g dw) α-Tocopherol 0.23 ± 0.02 3.37 ± 0.01 < 0.001 β-Tocopherol nd 0.22 ± 0.04 – δ-Tocopherol 0.09 ± 0.01 nd – Sum 0.33 ± 0.03 3.59 ± 0.05 < 0.001
dw– dry weight basis; nd – not detected. C8:0 – Caprylic acid; C10:0 – Capric acid; C12:0– Lauric acid; C14:0 – Myristic acid; C14:1 – Myristoleic acid; C15:0 – Pentadecanoic acid; C16:0 – Palmitic acid; C16:1 – Palmitoleic acid; C17:0 – Heptadecanoic acid; C18:0 – Stearic acid; C18:1n9 – Oleic acid; C18:2n6 – Linoleic acid; C18:3n3 – Linolenic acid; C20:0 – Arachidic acid; C20:2 – cis-11,14 - Eicosadienoic acid; C20:3n3– Eicosatrienoic acid; C22:0 – Behenic acid; C23:0– Tricosanoic acid; C24:0 – Lignoceric acid. SFA – saturated fatty acids, MUFA– monounsaturated fatty acids, PUFA – polyunsaturated fatty acids.
Table 3 Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data, tentative identi fi cation and quanti fi cation (mg/g dw) of the phenolic compounds present in fruits and stems of Lycium barbarum L. Peak Rt (min) λ max (nm) [M-H] − (m/z ) MS 2 (m/z ) Tentative identi fi cation Reference used for identi fi cation Fruits Stems t-Students test p -value 1 5.08 262 311 179(100),135(5) Caftaric acid A Mocan et al. (2015a , Mocan et al., 2015b 0.86 ± 0.04 nd – 2 5.37 313 353 191(100),179(7),161(3) cis 3-O -Ca ff eoylquinic acid A Cli ff ord et al. (2003 , 2005) 2.9 ± 0.1 0.36 ± 0.02 < 0.001 3 5.54 296 487 163(100),119(40) p -Coumaroyl acid dihexoside B Bondia-Pons et al., 2014 and Zhou et al. (2017) 3.6 ± 0.2 nd – 4 5.74 304 353 191(100),179(7),161(3) trans 3-O -Ca ff eoylquinic acid C Cli ff ord et al. (2003 , 2005) 8.87 ± 0.01 0.59 ± 0.02 < 0.001 5 5.82 264 343 191(3),169(100),125(3) Galloylquinic acid A Guimarães et al. (2013) nd 1.59 ± 0.01 – 6 7.16 324 933 609(100),301(5) Quercetin-dihexoside-rutinoside D Bondia-Pons et al. (2014) 3.73 ± 0.03 nd – 7 7.47 315 353 191(100),179(3),161(3) trans 5-O -Ca ff eoylquinic acid A DAD/MS, standard 3.3 ± 0.1 8.03 ± 0.01 < 0.001 8 7.92 284 385 223(100),207(50),179(40),163(14),149(3) Sinapic acid hexoside E Chahdoura et al. (2014) nd 2.8 ± 0.1 – 9 8.07 315 289 245(2),20(13),137(20) Catechin F DAD/MS, standard 10.4 ± 0.4 nd – 10 8.75 318 517 193(100), 179(5),149(20) Ferulic acid dihexoside G Dias et al. (2016) 0.9 ± 0.1 nd – 11 9.36 284 385 223(100),207(40),179(2),161(19),153(36),149(2) Sinapic acid hexoside E Chahdoura et al. (2014) nd 0.9 ± 0.1 – 12 10.36 322 179 161(5),159(4),135(100) Ca ff eic acid A DAD/MS, standard nd 0.52 ± 0.01 – 13 14.31 272 787 635(12),617(14),483(3),465(4),447(5),423(20),313(2),271(10) Tetragalloyl-glucose C Rached et al. (2016) nd 2.4 ± 0.1 – 14 15.79 310 163 119(100) p -Coumaric acid B DAD/MS, standard 12.3 ± 0.4 nd – 15 15.84 290 577 289(76),245(14),203(18) Procyanidin dimer F Pires et al. (2017) nd 6.2 ± 0.1 – 16 17.71 352 609 301(100) Quercetin-3-O -rutinoside (rutin) D DAD/MS, standard 16.6 ± 0.1 48 ± 1 – 17 18.42 nd 447 301(100) Quercetin-3-O -rhamnoside (quercitrin) H Mocan et al. (2014) , Protti et al. (2017) and Zhou et al. (2017) tr nd – 18 18.84 355 463 301(100) Quercetin-3-O -galactoside (hyperoside) H Qian et al. (2004) and Donno et al. (2015) 0.70 ± 0.01 nd – 19 19.11 353 463 301(100) Quercetin-3-O -glucoside (isoquercitrin) H DAD/MS, standard 2.42 ± 0.04 nd – 20 21.12 348 593 285(100) Kaempferol-3-O -rutinoside G DAD/MS, standard tr 0.83 ± 0.01 – 21 22.27 343 447 285(100) Kaempferol-3-O -glucoside G DAD/MS, standard 4.21 ± 0.04 nd – 22 27.41 nd 431 285(100) Kaempferol-rhamnoside G MS tr nd – Sum of phenolic acid 32.7 ± 0.8 17.2 ± 0.2 < 0.001 Sum of fl avan-3-ols 10.4 ± 0.4 6.2 ± 0.1 < 0.001 Sum of fl avonols 27.6 ± 0.1 48.5 ± 0.6 < 0.001 Sum of phenolic compounds 71 ± 1 71.9 ± 0.9 0.113 tr-trace amounts; nd-not detected. Standard calibration curves: A -ca ff eic acid (y = 406,369 + 388345 x , R 2= 0.9949); B -p -coumaric acid (y = 6966.7 + 301950 x , R 2= 0.9999); C -chlorogenic acid (y = – 161,172 + 168823 x , R 2= 0.9999); D - quercetin-3-O -rutinoside (y = 76,751 + 13343 x , R 2= 0.9998); E -sinapic acid (y = 30,036 + 197337 x , R 2= 0.9997); F -catequin (y = – 23,200 + 84950 x , R 2= 0.9999); G -ferulic acid (y = – 185,462 + 633126 x , R 2= 0.9999); H - quercetin-3-O -glucoside (y = – 160,173 + 34843 x , R 2= 0.9998); G - kaempferol-3-O -rutinoside (y = 30,861 + 11117 x ; R 2= 0.9998).
acid in the fruits of L. barbarum, although in lower amounts than the ones reported in this paper.
Peak14, identified as p-coumaric acid by comparison with a stan-dard, was the majority phenolic acid derivative in the fruits, whereas lower levels of caffeic acid (peak 12) were present in the stems. Other hydroxycinnamoyl derivatives detected in the samples were caftaric acid (peak1), previously described in the leaves of L. barbarum (Mocan et al., 2015a,b), and different glycosides (peaks 3, 8, 10 and 11). Peak 3 presented a pseudomolecular ion [M−H]−at m/z 487 releasing
frag-ments at m/z 163 (−324 mu, loss of two hexosyl moieties) and 119, which is coherent with a p-coumaroyl acid dihexoside, as reported in goji fruits from Finland and Spain (Bondia-Pons et al., 2014); 6-O-trans-p-coumaroyl-2-O-glucopyranosyl-a-D-glucopyranoside was also recently identified byZhou et al. (2017)in wolfberries from China. Similarly, peak10, with a pseudomolecular ion [M−H]−at m/z 517 and a main MS2product ion at m/z 193 from the loss of 324 mu, could be tenta-tively identified as a ferulic acid dihexoside. Peaks 8 and 11 presented the same pseudomolecular ion [M−H]−at m/z 385 and an MS2
frag-ment at m/z 223 (sinapic acid aglycone), corresponding to the loss of an
hexosyl unit, so that they were tentatively identified as sinapic acid hexosides.
Finally, peaks5 and 13 were associated to galloyl derivatives. The first one was identified as galloylquinic acid based on its pseudomole-cular ion ([M−H]−at m/z 343) and the major MS2fragment at m/z
169 [gallic acid-H]−, from the loss of quinic acid moiety (−152 mu). Peak13 was assigned as tetragalloyl-glucose from its pseudomolecular ion [M−H]−at m/z 787 and fragment ions at m/z 635, 483, and 313
from the consecutive loss of three gallic acid units. The identification of both compounds was supported by its comparison with previously ob-tained data available in a compound library (Guimarães et al., 2013; Rached et al., 2016). To the authors’ best knowledge, these compounds have not been previously cited in goji samples.
The total content of phenolic compounds did not show any statis-tically significant difference between fruits and stems of goji samples. However, significant differences were found between samples when considering each family of phenolic compounds, being phenolic acid derivatives the majority compounds in the fruits (32.7 mg/g vs 17.2 mg/g in the stems) and flavonols in the stems (48.5 mg/g vs Fig. 1. HPLC phenolic profile recorded at 280 nm of the hydromethanolic extracts of fruits (A) and stems (B) of L. barbarum. Peak numbering is according toTable 3.
Table 4
Antioxidant, hepatotoxic and antimicrobial activity of fruits and stems of Lycium barbarum L. (mean ± SD).
Fruits Stems t-Students test p-value Correlation factor r2
Phenolic acids Flavan-3-ols Flavonols Phenolic compounds Antioxidant activity EC50values (mg/mL)A
DPPH scavenging activity 6.25 ± 0.2 0.28 ± 0.02 < 0.001 0.998 0.996 0.999 0.880
Reducing power 1.09 ± 0.02 0.23 ± 0.01 < 0.001 0.999 0.997 0.999 0.880
β-carotene bleaching inhibition 1.9 ± 0.3 0.26 ± 0.02 < 0.001 0.973 0.971 0.974 0.857
TBARS inhibition 3.9 ± 0.2 0.07 ± 0.02 < 0.001 0.995 0.993 0.996 0.877
Hepatotoxicity GI50values (μg/mL)B
PLP2 > 400 > 400 – – – – –
Antimicrobial activity MIC values (mg/mL) Gram-negative bacteria
Acinetobacter baumannii > 20 20 – – – – –
Escherichia coli ESBL 1 5 5 – – – – –
Escherichia coli ESBL 2 5 10 – 0.999 0.997 0.999 0.880
Escherichia coli 2.5 2.5 – – – – –
Klebsiella pneumoniae 20 10 – 0.999 0.997 0.999 0.880
Klebsiella pneumoniae ESBL 20 20 – – – – –
Morganella morganii 5 5 – – – – – Pseudomonas aeruginosa 20 10 – 0.999 0.997 0.999 0.880 Gram-positive bacteria Enterococcus faecalis 2.5 10 – 0.999 0.997 0.999 0.880 Listeria monocytogenes 5 5 – – – – – Staphylococcus aureus 2.5 2.5 – – – – – MRSA 2.5 5 – 0.999 0.997 0.999 0.880 MSSA 2.5 10 – 0.999 0.997 0.999 0.880
EC50values correspond to the sample concentration achieving 50% of antioxidant activity or 0.5 of absorbance in reducing power assay. A- Trolox EC50values:
43.03 ± 1.71μg/mL (DDPH), 29.62 ± 3.15 μg/mL (reducing power), 2.63 ± 0.14 μg/mL (β-carotene bleaching inhibition) and 3.73 ± 1.9 μg/mL (TBARS in-hibition); B - Ellipticine GI50values: 2.29 mg/mL (PLP2). MIC values correspond to the minimal extract concentration that inhibited the bacterial growth. ESBL
27.6 mg/g in the fruits).
Quercetin-3-O-rutinoside was by far the major phenolic compound in stems (48 mg/g extract) and fruits (16.6 mg/g extract), followed in these latter by p-coumaric acid (12.3 mg/g extract). The differences between fruits and stems are explained by the clear difference in plant tissues. Although a greater amount offlavonols might be expected in the fruits, the obtained results could be explained by the edafoclimatic characteristics, degree of ripening and storage conditions (Haminiuk et al., 2012).
3.3. Bioactivities of fruit and stem hydromethanolic extracts
Data regarding the antioxidant, hepatotoxic and antibacterial ac-tivities are presented inTable 4. The hydromethanolic extracts of L. barbarum stems showed the highest antioxidant activity in all assays: DPPH scavenging activity, reducing power, β-carotene bleaching in-hibition and TBARS inin-hibition (EC50= 0.28, 0.23, 0.26, and 0.07 mg/
mL, respectively). Similar results were reported byLiu et al. (2017)in ethanolic extracts of L. barbarum stems from Taiwan, namely DPPH scavenging activity and reducing power (0.102 and 0.167 mg/mL, re-spectively). On the other hand,Jabbar et al. (2014)reported lower EC50
values in methanolic extracts of goji fruits from China, regarding DPPH scavenging activity and reducing power (0.042 and 0.121 mg/mL, re-spectively), in comparison with the herein analysed hydromethanolic extract.
The antioxidant activity revealed by the herein study samples could be due to their high content in phenolic acids derivatives and flavo-noids. The antioxidant activity of phenolic acid derivatives depends on the number of hydroxyl groups in the molecule, that would be strengthened by steric hindrance. Moreover, the electron-withdrawing properties of the carboxylate group in benzoic acids has a negative influence on the H-donating abilities of the hydroxy benzoates, being hydroxylated cinnamates more effective than benzoate counterpart (Rice-Evans et al., 1996). The presence of multiple hydroxyl groups in flavonoids and other phenolics structures gives them a reducing char-acter. In fact, it has been shown in in vitro assays that many of these compounds possess a strong antioxidant activity. This activity is parti-cularly high, three to four fold higher in ortho-dihydroxyflavonoids (those containing a catechol group in their aromatic rings) such as flavonols or flavanol (Vicente and Boscaiu, 2018). Thus, the differences in the phenolic compounds family present in each of the study plant part (phenolic acids for fruits andflavonols in stems), could explain the greater antioxidant capacity of the stems.
Neither fruits nor stems revealed toxicity towards a porcine liver primary culture (PLP2).
Regarding antibacterial activity (Table 4), both samples showed better results against Gram-positive than against Gram-negative bac-teria, with MIC values ranging between 2.5 and 10 mg/mL. The lowest MIC values were determined for E. faecalis (2.5 mg/mL), L. mono-cytogenes (5 mg/mL), S. aureus (2.5 mg/mL), MRSA (2.5 mg/mL), and MSSA (2.5 mg/mL). As for Gram-negative bacteria, the stems presented higher activity against A. baumannii (20 mg/mL), K. pneumonia (10 mg/ mL), and P. aeruginosa (10 mg/mL).Mocan et al. (2017) andMocan et al. (2015b)reported lower MIC values in goji leaves andflowers, respectively, against both Gram-negative and positive bacteria. A pos-sible explanation could be that the bacteria used by those authors were ATCC (with no resistance profile), so that lower concentrations of ex-tracts could be needed to inhibit the growth of the bacterial strains.
As it can also be seen inTable 4good correlations were obtained between the different families of analysed phenolic compounds and the antioxidant activity (r2< 0.8). Regarding antibacterial assays, good
correlation coefficients were observed for Escherichia coli ESBL 2, Klebsiella pneumonia, Pseudomonas aeruginosa, and Enterococcus faecalis. The presence of phenolic compounds, namelyflavonoids and phe-nolic acids (e.g. chlorogenic acids derivatives) could be related to the antibacterial potential of the study samples. Flavonoids are known to be
synthesized by plants in response to microbial infection, thus explaining the in vitro antimicrobial activity of these substances against a wide array of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins, which may disrupt the microbial membrane (Cushnie and Lamb, 2005;Kabir et al., 2014). The antimicrobial activity of polyphenols has also been attributed to their structural features, as well as pH and sodium chloride con-centration, resulting in physiological changes in the microorganisms and eventual cell death (Kabir et al., 2014). Chlorogenic acid is a phenolic ester of caffeic acid and (−)-quinic acid (Chiang et al., 2004), which is metabolized into active compounds, such as quinic, caffeic, benzoic, hippuric, ferulic, isoferulic, and hydroxybenzoic acids. Studies carried out byKabir et al. (2014)confirm that chlorogenic acids and related compounds exhibited a potent antimicrobial activity, and a synergistic effect between compounds. Thus, these compounds could be related to the antimicrobial potential revealed in these plant parts.
Overall, the stems of L. barbarum showed higher values of energy, fat, proteins and ash, as also monounsaturated fatty acids, tocopherols, and flavonols (i.e., quercetin-3-O-rutinoside). They also presented greater antioxidant capacity and higher activity against Gram-negative bacteria. The fruits of L. barbarum possessed higher contents of sugars (mainly fructose and glucose), as expected, polyunsaturated fatty acids, hydroxycinnamoyl derivatives (p-coumaric acid and chlorogenic acid derivatives), andflavan-3-ols (catechin); they also showed higher ac-tivity against Gram-positive bacteria.
All in all, this study allowed verifying that not only the fruit but also goji stems can be sources of compounds with interesting nutritional and bioactive properties and, therefore, they could be useful for nu-traceutical formulations or its incorporation into foods with functional properties. Since stems are by-products, besides its possible beneficial effects to consumers, they also provide industrial sustainability and could be used as an add value by-product scarcely noticed up to now. Acknowledgements
The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), T.C.S.P. Pires (SFRH/BD/129551/2017) grant and L. Barros contract. The GIP-USAL isfinancially supported by the Spanish Government through the project AGL2015-64522-C2-2-R. The authors are grateful to FEDER-Interreg España-Portugal programme forfinancial support through the project 0377_Iberphenol_6_E.
Appendix A. Supplementary data
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